This study was designed to demonstrate that differentiation of stomach cancer cells can be modified by microenvironmental change and to look for a method inducing or promoting tumor cell differentiation. To evaluate the biomorphological characterization of tumor cell differentiation in suramin-containing in vitro culture of human stomach cancer cell lines, inverted phase-contrast microscopic examination, analysis of growth curves and BrdU-positive S-phase fraction, immunocytochemical study, radioimmunoassay for CEA, transmission electron microscopic examination, DNA flow cytometry, and heterotransplantation in SCID mice were performed. Suramin inhibited tumor cell growth. Development of intracytoplasmic lumina and intercellular lumina was noted in suramin-containing culture with formation of numerous microvilli and frequent desmosomes. The amount of CEA released by a cell was increased in suramin-containing culture. Suramin inhibited heterotransplantation, and a transplant from suramin-containing culture revealed a much higher degree of differentiation than that from suramin-absent culture. Suramin induced no change in DNA ploidy pattern. Elimination of suramin from the culture medium did not reverse the tumor cell differentiation. Each stomach cancer cell line showed a different degree of responsiveness to suramin. In conclusion, this study shows that suramin inhibits growth of SNU-5 and SNU-16 cells and that suramin induces differentiation of SNU-16 cells.